Reconstitution of a fungal meroterpenoid biosynthesis reveals the involvement of a novel family of terpene cyclases

Journal name:
Nature Chemistry
Volume:
2,
Pages:
858–864
Year published:
DOI:
doi:10.1038/nchem.764
Received
Accepted
Published online

Abstract

Meroterpenoids are hybrid natural products of both terpenoid and polyketide origin. We identified a biosynthetic gene cluster that is responsible for the production of the meroterpenoid pyripyropene in the fungus Aspergillus fumigatus through reconstituted biosynthesis of up to five steps in a heterologous fungal expression system. The cluster revealed a previously unknown terpene cyclase with an unusual sequence and protein primary structure. The wide occurrence of this sequence in other meroterpenoid and indole–diterpene biosynthetic gene clusters indicates the involvement of these enzymes in the biosynthesis of various terpenoid-bearing metabolites produced by fungi and bacteria. In addition, a novel polyketide synthase that incorporated nicotinyl-CoA as the starter unit and a prenyltransferase, similar to that in ubiquinone biosynthesis, was found to be involved in the pyripyropene biosynthesis. The successful production of a pyripyropene analogue illustrates the catalytic versatility of these enzymes for the production of novel analogues with useful biological activities.

At a glance

Figures

  1. Representative fungal meroterpenoids and their biological activities.
    Figure 1: Representative fungal meroterpenoids and their biological activities.

    The terpenoid part is shown in blue and the polyketide part in red. The disease states for which these biological activities are thought to be important are also given. Ac = acetyl.

  2. Proposed biosynthetic pathway of pyripyropene A (9) in A. fumigatus.
    Figure 2: Proposed biosynthetic pathway of pyripyropene A (9) in A. fumigatus.

    A nicotinic acid (1)-derived CoA (2) is condensed with two molecules of malonyl-CoA to form a pyrone 3 through a polyketide pathway. A terpenoid unit 4 is attached and, after epoxidation and cyclization, produces the core structure of pyripyropene (7). PP = diphosphate; PKS = polyketide synthase; PT = prenyltransferase; FMO = (FAD)-dependent monooxygenase.

  3. The pyripyropene biosynthetic gene cluster (pyr cluster) identified from A. fumigatus.
    Figure 3: The pyripyropene biosynthetic gene cluster (pyr cluster) identified from A. fumigatus.

    a, Deduced functions of each open reading frame and their amino acid sequence similarity to and identity with other known proteins are shown (genes studied herein and their functions identified are coloured). b, Schematic representation of the cluster. The direction of the arrow indicates the direction from the start to the stop codon.

  4. Products isolated from coexpression experiments.
    Figure 4: Products isolated from coexpression experiments.

    a, HPLC profile of a culture media from each A. oryzae transformant: i, control transformant that harbours a void vector; ii, transformant that expresses only Pyr2; iii, transformant that harbours pyr1 and pyr2 genes. Coexpression of the CoA ligase and the PKS genes resulted in the production of the polyketide pyrone 3. Chromatograms monitored at 254 nm absorption in absorbance units (AU). b, HPLC profile of the mycelial extracts of the transformant cultures: i, transformant that harbours pyr1, pyr2 and pyr6; ii, transformant that harbours pyr6 and pyr5 fed with 3; iii, transformant that harbours pyr6, pyr5 and pyr4 fed with 3. The cyclized product 7 was obtained only when the FMO and cyclase genes were coexpressed. Chromatograms monitored at 322 nm. Absorption in milliabsorbance units (mAU).

  5. Functional analyses of Pyr4 in vitro.
    Figure 5: Functional analyses of Pyr4 in vitro.

    a, HPLC profile of in vitro reaction products of Pyr4 with 8: i, substrate with assay buffer; ii, substrate with microsomal proteins from control transformant that harboured a void vector; iii, substrate with soluble proteins from transformant that harboured pyr4; iv, substrate with microsomal proteins from transformant that harboured pyr4; v, authentic deacetyl-pyripyropene E (7). Pyr4 was shown to be a stand-alone terpene cyclase. b, HPLC profile of yeast in vitro reaction products of Pyr4 mutants with 8: i, wild-type Pyr4; ii, E63Q mutant; iii, E63A mutant; iv, D218N mutant; v, D218A mutant; vi, void-vector control. Chromatograms from mutants are indicated in green and the wild type (WT) in red. A small peak that appears at around the same retention time as 7 in (ii)–(vi) is unrelated to reaction with 8. Both Glu63 and Asp218 are shown to be catalytically important residues.

  6. A novel meroterpenoid (12) formed through HPhPO (11) from benzoic acid by the pyripyropene biosynthetic machinery.
    Figure 6: A novel meroterpenoid (12) formed through HPhPO (11) from benzoic acid by the pyripyropene biosynthetic machinery.

    Incorporation of a phenyl ring in the structure was achieved by replacing nicotinic with benzoic acid.

Compounds

12 compounds View all compounds
  1. Nicotinic acid
    Compound 1 Nicotinic acid
  2. (2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-hydroxy-2-(((((((R)-3-hydroxy-2,2-dimethyl-4-((3-((2-(nicotinoylthio)ethyl)amino)-3-oxopropyl)amino)-4-oxobutoxy)oxidophosphoryl)oxy)oxidophosphoryl)oxy)methyl)tetrahydrofuran-3-yl phosphate
    Compound 2 (2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-hydroxy-2-(((((((R)-3-hydroxy-2,2-dimethyl-4-((3-((2-(nicotinoylthio)ethyl)amino)-3-oxopropyl)amino)-4-oxobutoxy)oxidophosphoryl)oxy)oxidophosphoryl)oxy)methyl)tetrahydrofuran-3-yl phosphate
  3. 4-Hydroxy-6-(pyridin-3-yl)-2H-pyran-2-one
    Compound 3 4-Hydroxy-6-(pyridin-3-yl)-2H-pyran-2-one
  4. (2E,6E)-3,7,11-Trimethyldodeca-2,6,10-trien-1-yl diphosphate
    Compound 4 (2E,6E)-3,7,11-Trimethyldodeca-2,6,10-trien-1-yl diphosphate
  5. 4-Hydroxy-6-(pyridin-3-yl)-3-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)-2H-pyran-2-one
    Compound 5 4-Hydroxy-6-(pyridin-3-yl)-3-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)-2H-pyran-2-one
  6. 3-((2E,6E)-10,11-Dihydroxy-3,7,11-trimethyldodeca-2,6-dien-1-yl)-4-hydroxy-6-(pyridin-3-yl)-2H-pyran-2-one
    Compound 6 3-((2E,6E)-10,11-Dihydroxy-3,7,11-trimethyldodeca-2,6-dien-1-yl)-4-hydroxy-6-(pyridin-3-yl)-2H-pyran-2-one
  7. (3S,4aR,6aR,12aR,12bS)-3-Hydroxy-4,4,6a,12b-tetramethyl-9-(pyridin-3-yl)-1,3,4,4a,5,6,6a,12,12a,12b-decahydrobenzo[f]pyrano[4,3-b]chromen-11(2H)-one
    Compound 7 (3S,4aR,6aR,12aR,12bS)-3-Hydroxy-4,4,6a,12b-tetramethyl-9-(pyridin-3-yl)-1,3,4,4a,5,6,6a,12,12a,12b-decahydrobenzo[f]pyrano[4,3-b]chromen-11(2H)-one
  8. 3-((2E,6E)-9-((S)-3,3-Dimethyloxiran-2-yl)-3,7-dimethylnona-2,6-dien-1-yl)-4-hydroxy-6-(pyridin-3-yl)-2H-pyran-2-one
    Compound 8 3-((2E,6E)-9-((S)-3,3-Dimethyloxiran-2-yl)-3,7-dimethylnona-2,6-dien-1-yl)-4-hydroxy-6-(pyridin-3-yl)-2H-pyran-2-one
  9. (3S,4R,4aR,6S,6aS,12R,12aS,12bS)-4-(Acetoxymethyl)-12-hydroxy-4,6a,12b-trimethyl-11-oxo-9-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,11,12,12a,12b-dodecahydrobenzo[f]pyrano[4,3-b]chromene-3,6-diyl diacetate
    Compound 9 (3S,4R,4aR,6S,6aS,12R,12aS,12bS)-4-(Acetoxymethyl)-12-hydroxy-4,6a,12b-trimethyl-11-oxo-9-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,11,12,12a,12b-dodecahydrobenzo[f]pyrano[4,3-b]chromene-3,6-diyl diacetate
  10. Benzoic acid
    Compound 10 Benzoic acid
  11. 4-Hydroxy-6-phenyl-2H-pyran-2-one
    Compound 11 4-Hydroxy-6-phenyl-2H-pyran-2-one
  12. (3S,4aR,6aR,12aR,12bS)-3-Hydroxy-4,4,6a,12b-tetramethyl-9-phenyl-1,3,4,4a,5,6,6a,12,12a,12b-decahydrobenzo[f]pyrano[4,3-b]chromen-11(2H)-one
    Compound 12 (3S,4aR,6aR,12aR,12bS)-3-Hydroxy-4,4,6a,12b-tetramethyl-9-phenyl-1,3,4,4a,5,6,6a,12,12a,12b-decahydrobenzo[f]pyrano[4,3-b]chromen-11(2H)-one

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Affiliations

  1. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan

    • Takayuki Itoh,
    • Kinya Tokunaga,
    • Yudai Matsuda,
    • Ikuro Abe,
    • Yutaka Ebizuka &
    • Tetsuo Kushiro
  2. School of Pharmacy, Iwate Medical University, Nishitokuta, Yahaba, Iwate, Japan

    • Isao Fujii
  3. Present address: School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan

    • Tetsuo Kushiro

Contributions

T.I., Y.E. and T.K. conceived and designed the experiments, T.I., K.T. and Y.M. performed the experiments, T.I., K.T., Y.M., I.A., Y.E. and T.K. analysed the data, I.F. contributed the fungal expression system and T.I., Y.E. and T.K. co-wrote the paper.

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The authors declare no competing financial interests.

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